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Role of iron in brain lipocalin 2 upregulation after intracerebral hemorrhage in rats
brain research 1505 (2013) 86–92
Available online at www.sciencedirect.com
www.elsevier.com/locate/brainres
Research Report
Role of iron in brain lipocalin 2 upregulation after intracerebral hemorrhage in rats Ming Donga,b, Guohua Xia, Richard F. Keepa, Ya Huaa,n a
Deparment of Neurosurgery, University of Michigan, Ann Arbor, MI, USA Department of Neurology, 1st Affiliated Hospital, Jilin University, Changchun, China
b
ar t ic l e in f o
abs tra ct
Article history:
Brain iron overload has a detrimental role in brain injury after intracerebral hemorrhage
Accepted 6 February 2013
(ICH). Lipocalin 2 (LCN2), a siderophore-binding protein, is involved in cellular iron
Available online 15 February 2013
transport. The present study investigated changes in LCN2 expression after ICH and the
Keywords:
role of iron in those changes. Male Sprague-Dawley rats had an intracaudate injection of
Cerebral hemorrhage
autologous blood (ICH) or iron. Control rats received a needle insertion or saline injection.
Lipocalin 2
Some ICH animals were treated with either vehicle or deferoxamine, an iron chelator. Brain
Iron
LCN2 expression was determined by Western blot analysis and immunohistochemistry.
Deferoxamine
Real-time PCR was also used to confirm brain LCN2 mRNA expression. The number of
Immunohistochemistry
LCN2 positive cells was markedly increased in the ipsilateral basal ganglia and cortex after ICH and most LCN2 positive cells were astrocytes. Western blots showed that brain LCN2 levels were higher at days 1, 3 and 7 in the ipsilateral hemisphere after ICH (70 to 80 fold higher than contralateral hemisphere or sham-operated rats at 3 days), and declined to lower levels at day 14. Iron, but not saline injection also caused brain LCN2 upregulation (a more than 100-fold increase). In addition, systemic treatment of deferoxamine reduced ICH-induced LCN2 upregulation (po0.05). These results suggest that iron has a role in brain LCN2 upregulation following ICH. LCN2 upregulation after ICH may be part of the response to clear iron released from the hematoma during clot resolution. & 2013 Elsevier B.V. All rights reserved.
1.
Introduction
Intracerebral hemorrhage (ICH) is a devastating form of stroke with high morbidity and mortality (Qureshi et al., 2009; Rincon and Mayer, 2012). Evidence suggests that ironoverload is involved in ICH-induced brain damage resulting in perihematoma edema, neuronal death and brain atrophy (Keep et al., 2012; Xi et al., 2006). We have demonstrated that
deferoxamine, an iron chelator, reduces ICH-induced brain injury in young and aged rats (Okauchi et al., 2010; Xi et al., 2006), as well as in pigs (Gu et al., 2009). Clinical studies also found that high levels of serum ferritin, an iron storage protein, are independently associated with severe brain edema and poor prognosis in ICH patients (Mehdiratta et al., 2008; Perez de la Ossa et al., 2010), suggesting a role of iron in ICH-induced brain injury (Selim et al., 2011).
Abbreviations: ICH, Intracerebral hemorrhage; LCN2, Lipocalin 2; GFAP, Glial acidic fibrillary protein n Correspondence to: R5018 Biomedical Science Research Building, University of Michigan, 109 Zina Pitcher Place, Ann Arbor, MI 48109-2200, USA. Fax: þ1 734 763 7322. E-mail address: [email protected] (Y. Hua). 0006-8993/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.brainres.2013.02.008
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brain research 1505 (2013) 86–92
Lipocalin 2 (LCN2), also known as neutrophil gelatinase associated lipocalin (NGAL) or 24p3, is a siderophore-binding protein. LCN2 mediates iron uptake in cells expressing the LCN2 cell surface receptor, LCN2R (24p3R) (Devireddy et al., 2005). Recently, the function of LCN2 within the CNS has started to be elucidated. In vivo studies have demonstrated LCN2 expression in the choroid plexus in response to peripheral lipopolysaccharide administration (Marques et al., 2008) and excitotoxic brain injury (Chia et al., 2011). Recent studies showed that LCN2 plays a detrimental role in the CNS after spinal cord injury (Rathore et al., 2011) but it appears to play a protective role in experimental autoimmune encephalomyelitis (Berard et al., 2012). In the present study, we examined the time course of brain LCN2 expression in a rat model of ICH. The role of iron in ICH-induced LCN2 expression was also examined.
2.
Results
ICH-induced changes in LCN2 were measured by immunohistochemistry and Western blotting. LCN2 positive cells were found in the ipsilateral basal ganglia but not the contralateral basal ganglia at days 1, 3, 7 and 14 after ICH (Fig. 1A). The LCN2 protein levels determined by Western blotting in the ipsilateral basal ganglia were significantly increased at day 1, remained at high levels at days 3 and 7, and the decreased markedly at day 14 after ICH (Fig. 1B). Similar results were found in the ipsilateral cortex following ICH (Fig. 1C). LCN2 immunoreactivity was very weak in sham-operated brain but increased significantly after blood injection. LCN2 positive cells were astrocyte-like (Fig. 2A). Western blots, with
day 1
LCN2 normalized to b-actin, indicated that LCN2 protein levels in the ipsilateral basal ganglia were 71-fold higher than those in the contralateral basal ganglia (LCN2/b-actin ratio: 2.7070.46 vs. 0.0470.01, po0.001) and 84-fold higher than in sham-operated animals (0.9270.14 vs. 0.0170.01, po0.001) at 3 days after ICH (Fig. 2B). Similar results were also found in the cortex (Fig. 2B). Double-labeling was used to determine which types of cells express LCN2 after ICH. We found that LCN2 positive cells predominantly colocalized with glial fibrillary acid protein (GFAP) positive cells after ICH. There were only a few LCN2 positive cells that colocalized with neuronal nuclear antigen (NeuN) in cortex (Fig. 3). These results suggest that LCN2 is mainly expressed in astrocytes following ICH. To test whether iron can induce LCN2 expression, 30 mL of ferrous chloride (1 mmol/L) or saline was injected into the rats’ right basal ganglia. There were many more LCN2 positive cells in the ipsilateral basal ganglia after iron injection compared to saline injection (Fig. 4A). Iron injection caused a 136-fold increase of LCN2 protein levels at day 3 (ratio of LCN2/b-actin: 1.7770.43 vs. 0.01370.004 in saline injection, po0.01, Fig. 4B). To examine whether ICH-induced LCN2 expression can be attenuated by iron chelation, rats were treated with deferoxamine or vehicle (2 h and then every 12 h for 7 days) after ICH. Deferoxamine reduced ICH-induced LCN2 upregulation at day 7 (ratio of LCN2/b-actin: 0.4470.23 vs. 1.7370.5 in vehicle, po0.05, Fig. 5). To further elucidate whether the brain produces LCN2 after ICH, quantitative real-time PCR of LCN2 gene expression was examined in triplicate using Eppendorf Mastercycler Realplex Detection System. Real-time PCR showed that LCN2 mRNA expression in the ipsilateral basal ganglia was increased 6.5-fold compared to the contralateral basal ganglia at 24 h after ICH (po0.05).
day 3
day 7
day 14
Ipsilateral basal ganglia * Contralateral basal ganglia
Ipsilateral basal ganglia day1
day3
day7
Ipsilateral cortex day1
day14
LCN2
LCN2
β−actin
β−actin
day3
day7
day14
Fig. 1 – Time course showing LCN2 immunoreactivity in the ipsi- and contra-lateral basal ganglia (A), and the protein levels of LCN2 and b-actin in the ipsilateral basal ganglia (B) and cortex (C) after an injection of 100 ll autoglous blood into the right caudate. The asterisk indicates the hematoma, the boxes indicate the locations where higher powered immunohistochemistry images were taken. Scale bar¼ 100 lm.
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brain research 1505 (2013) 86–92
ICH Sham
Contraleteral
Ipsilateral
Basal ganglia
Cortex
Bar =100μm
ICH Basal ganglia
Ipsilateral
ICH
Sham
ICH
Sham
LCN2
LCN2
β−actin
β−actin ICH Ipsilateral
Cortex
Contralateral
Contralateral
LCN2
LCN2
β−actin
β−actin
Fig. 2 – LCN2 immunoreactivity in the ipsilateral basal ganglia and cortex after a sham operation, and in the contra- and ipsilateral basal ganglia and cortex after ICH at day 3(A), scale bar¼ 100 lm. The protein levels of LCN2 and b-actin determined by Western blotting in the basal ganglia and cortex (B) 3 days after sham operation or blood injection.
LCN2
GFAP
Merge
LCN2
NeuN
Merge
Fig. 3 – LCN2, glial fibrillary acidic protein (GFAP) and NeuN immunoreactivity and the colocalization of LCN2 with GFAP or NeuN by double labeling in the ipsilateral basal ganglia at day 3 after 100 ll blood infused into the right basal ganglia. Scale bar¼ 50 lm.
3.
Discussion
The major findings of the present study are: (1) brain LCN2 levels are markedly increased after ICH; (2) most LCN2 positive cells after ICH are astrocytes; (3) iron can upregulate brain LCN2; and (4) deferoxamine reduces ICH-induced LCN2
upregulation. These findings suggest an important role of iron in brain LCN2 upregulation after ICH and potentially a role of LCN2 in handling iron that is released from the hematoma after ICH. LCN2 expression in the brain is prominently increased at an early stage of ICH, which may be associated with acute
brain research 1505 (2013) 86–92
89
Iron injection
Saline injection
Basal ganglia
Cortex
Saline injection
Iron injection
Basal ganglia
β−actin
Fig. 4 – LCN2 immunoreactivity in the ipsilateral basal ganglia and cortex, and the protein levels of LCN2 and b-actin in the ipsilateral basal ganglia at day 3 after 30 ll of ferrous iron (1.0 mmol/L) or saline injection into the rat basal ganglia. Scale bar¼ 100 lm.
ICH+Vehicle
ICH+DFX
LCN2
ICH+Vehicle
ICH+DFX
LCN2
β-actin
Fig. 5 – (A) LCN2 immunohistochemistry of the ipsilateral basal ganglia, and (B) Western blot of that tissue from rats treated with deferoxamine (DFX) or vehicle for 7 days after 100 ll blood was injected into the right caudate. Scale bar¼100 lm.
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brain injury. There was a very marked increase in LCN2 in the perihematomal zone of basal ganglion and cortex occurs 1 day after ICH. Brain LCN2 levels were high for 7 days and decreased around 14 days after ICH. Although levels of LCN2 mRNA and protein expression are low in most regions of the normal brain (Chia et al., 2011), LCN2 is rapidly inducible (Liu and Nilsen-Hamilton, 1995). Recent work showed that LCN2 expression can be induced in the central nervous system following injury and neuroinflammation (Berard et al., 2012; Chia et al., 2011; Ip et al., 2011). Our study also confirmed that the brain can produce LCN2. Many factors may cause brain LCN2 upregulation after ICH. Our results showed that intracerebral injection of iron increases brain LCN2 levels and deferoxamine, an iron chelator, reduces ICH-induced upregulation of brain LCN2. These results indicate a role of iron in regulating LCN2 expression following ICH. However, LCN2 is an acute phase protein and other factors, including tumor necrosis factoralpha (TNF-a) and thrombin, two major factors which cause brain injury after ICH (Bodmer et al., 2012), may also upregulate brain LCN2 in the acute phase. For example, TNF-a is increased in the brain two hours after ICH (Hua et al., 2006) and TNF-a can upregulate LCN2 (Naude et al., 2012). Future studies should determine whether thrombin has a role in ICH-induced upregulation of brain LCN2. The role of LCN2 in ICH is unclear, but LCN2 upregulation in the brain after ICH may affect iron homeostasis. It is well known that iron overload following ICH causes brain damage. LCN2 transfers iron from intracellular to the extracellular medium via binding with intracellular siderophore, thereby reducing intracellular iron concentration. A report has suggested that LCN2 could be a mediator of an alternative, transferrin-independent pathway for cellular iron delivery (Yang et al., 2002). The iron chelator, deferoxamine, protects endotoxin-induced toxicity and mortality in LCN2 deficient mice (Srinivasan et al., 2012). Future studies should determine whether LCN2 can enhance brain iron clearance following ICH. We observed that LCN2 was mainly expressed in astrocytes after ICH. Our results are supported by other studies. In the case of kainite-induced brain injury, LCN2 is highly expressed in the reactive astrocytes in the injured hippocampus (Chia et al., 2011). Similarly, after spinal cord contusion injury, Rathore et al. (2011) found a strong induction of LCN2 in astrocytes but not microglia. After ICH, the breakdown of hemoglobin in the hematoma results in the release of iron. Thus, there is an increase in perihematomal iron, that can be detected by Perls’ staining, which persists for weeks and is mostly present in glial cells (Wu et al., 2003). LCN2 upregulation in perihematomal astrocytes suggests that it may be involved in, or a response to, iron accumulation. We also found some expression of LCN2 in neurons after ICH. LCN2 is also found to be expressed in neurons and upregulated in the hippocampus by stress (Mucha et al., 2011). Neuronal expression of LCN2 has been observed following exposure to trauma (Rathore et al., 2011) or neuroinflammation (Berard et al., 2012; Chia et al., 2011; Ip et al., 2011). After ICH, Perls’ staining indicates that there is a build-up of iron in some neurons (Wu et al., 2003). Whether LCN2 participates in that uptake and in the neuronal injury that follows ICH deserves further investigation.
In conclusion, LCN2 is markedly upregulated in the brain after ICH. As an intracerebral iron injection induces brain LCN2 and iron chelation reduces ICH-induced LCN2 upregulation, iron appears to play a central role in the LCN2 response to hemorrhage. LCN2 may be involved in iron homeostasis after cerebral hemorrhage and modifying brain LCN2 levels may affect ICH-induced brain iron overload and brain injury.
4.
Experimental procedures
4.1.
Animal preparation and intracerebral injection
Animal use protocols were approved by the University of Michigan Committee on the Use and Care of Animals. A total of 96 male Sprague-Dawley rats (weighed 275–350 g, Charles River Laboratories, Portage, MI, USA) were used in these studies. Animals were housed under standard 12:12 h light–dark conditions and allowed free access to food and water. Animals were anesthetized with pentobarbital (45 mg/kg, i.p.). Septic precautions were used in all surgical procedures and rectal temperature was maintained at 37.5 1C using a feedback-controlled heating pad. A polyethylene catheter (PE-50) was inserted into the right femoral artery to monitor arterial blood pressure and blood gases and to obtain blood for intracerebral infusion. The animals were positioned in a stereotactic frame (Kopf Instruments) and a cranial burr hole (1 mm) was drilled. Rats received an autologous blood injection into the right basal ganglia (coordinates: 0.2 mm anterior, 3.5 mm lateral, and 5.5 mm ventral to the bregma) through a 26 g needle at a rate of 10 mL/min using a microinfusion pump.
4.2.
Experimental groups
This study was divided into four parts. In the first part, rats received either an intracerebral injection of 100 mL autologous whole blood or a needle insertion (sham). Rats were euthanized 1, 3, 7, or 14 days later for Western blot analysis (n¼ 4 for each time point) and immunohistochemistry (n¼ 4 for each time point). In the second part, rats had an infusion of 30 mL ferrous chloride (1 mmol/L) or saline. Rat brains were sampled at 3 days after intracerebral injection and used for Western blot analysis (n ¼4) and immunohistochemistry (n ¼4). In the third part, rats had an intracerebral infusion of 100 mL autologous whole blood and were treated with either deferoxamine (100 mg/kg, i.p. at 2 h after ICH and then every 12 h for 7 days) or vehicle. Rats were euthanized at day 7 for Western blot analysis (n¼ 4) and immunohistochemistry (n ¼4). In the fourth part, rats had a 100 ml blood injection in the right basal ganglia and were killed at 24 h for RNA isolation and real-time PCR analysis.
4.3.
Western blot analysis
Western blot analysis was performed as previously described (Xi et al., 1999). Briefly, brain tissue was immersed in Western sample buffer and sonicated. Protein concentration was determined by Bio-Rad protein assay kit and 50 mg protein samples were separated by sodium dodecyl sulfate–polyacrylamide gel
brain research 1505 (2013) 86–92
electrophoresis and transferred to a Hybond-C pure nitrocellulose membrane (Amersham). Membranes were probed with primary antibody: goat anti-lipocalin 2 antibody (1:200 dilution, R&D Systems). The secondary antibody was rabbit anti-goat IgG (Bio-Rad; 1:2500 dilution). The antigen–antibody complexes were visualized with the ECL chemiluminescence system (Amersham) and exposed to Kodak X-OMAT film. The relative densities of bands were analyzed with NIH ImageJ.
4.7.
91
Statistical analysis
All data in this study are presented as mean7SD. Data were analyzed with Student’s t-test and analysis of variance. The P value of less than 0.05 was considered as significant level.
Acknowledgment 4.4.
Immunohistochemistry
Immunohistochemistry was performed as described previously (Xi et al., 1999). Briefly, rats were anesthetized and subjected to intracardiac perfusion with 4% paraformaldehyde in 0.1 mol/L phosphate-buffered saline (pH 7.4). Brains were removed and kept in 4% paraformaldehyde for 6 h, then immersed in 30% sucrose for 3–4 days at 4 1C. After embedding in a mixture of 30% sucrose and OCT (SAKURA Finetek, USA), 18 mm sections were taken on a cryostat. Immunohistochemistry staining was then performed using the avidin–biotin complex technique. The primary antibody was goat anti-lipocalin 2 antibody (1:200 dilution, B&D Systems). The secondary antibody was rabbit anti-goat IgG (Bio-Rad; 1:2500 dilution).
4.5.
Immunofluorescent double-labeling
For immunofluorescent double-labeling, the primary antibodies were goat anti-lipocalin 2 (1:200 dilution, R&D Systems), mouse anti-NeuN (1:200 dilution, Millipore), and mouse antiGFAP (1:200 dilution, Millipore). Rhodamine-conjugated donkey anti-goat (Invitrogen; 1:500 dilution), fluorescein isothiocyanate (FITC)-labeled horse anti-mouse (Vector; 1:500 dilution) and donkey anti-rabbit antibodies were used as secondary antibodies. The double-labeling was analyzed using a fluorescence microscope (Xi et al., 1999).
4.6. Quantitative real-time polymerase chain reaction (qPCR) Total RNA was prepared from 20–30 mg the ipsilateral basal ganglia tissue using an RNeasy Mini kit (Qiagen), and stored at 80 1C. 2 mg mRNA was used for cDNA synthesis using High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA). Quantitative real-time PCR of gene expression were run in triplicate using TaqMan PCR Master Mix (Roche). No template controls were prepared by omitting to add cDNA template to the reaction. Real-time quantitative primers were designed and purchased from Applied Biosystems (Life Technologies Corporation). The rat LCN2 primers were 50 -GAT TCG TCA GCT TTG CCA AGT-30 (forward primer) and 50 -CAT TGG TCG GTG GGA ACA G-30 (reverse primer). A positive standard curve for each primer was obtained using serially-diluted cDNA sample mixture. The quantity of gene expression was calculated using standard samples and normalized with GAPDH.
This study was supported by Grants NS-039866, NS-057539, NS-073595 and NS-079157 from the National Institutes of Health (NIH). The content is solely the responsibility of the authors and does not necessarily represent official views of the NIH.
r e f e r e nc e s
Berard, J.L., Zarruk, J.G., Arbour, N., Prat, A., Yong, V.W., Jacques, F.H., Akira, S., David, S., 2012. Lipocalin 2 is a novel immune mediator of experimental autoimmune encephalomyelitis pathogenesis and is modulated in multiple sclerosis. Glia 60, 1145–1159. Bodmer, D., Vaughan, K.A., Zacharia, B.E., Hickman, Z.L., Connolly Jr., E.S., 2012. The molecular mechanisms that promote edema after intracerebral hemorrhage. Transl. Stroke Res. 3, S52–S61. Chia, W.J., Dawe, G.S., Ong, W.Y., 2011. Expression and localization of the iron-siderophore binding protein lipocalin 2 in the normal rat brain and after kainate-induced excitotoxicity. Neurochem. Int. 59, 591–599. Devireddy, L.R., Gazin, C., Zhu, X., Green, M.R., 2005. A cellsurface receptor for lipocalin 24p3 selectively mediates apoptosis and iron uptake. Cell 123, 1293–1305. Gu, Y., Hua, Y., Keep, R.F., Morgenstern, L.B., Xi, G., 2009. Deferoxamine reduces intracerebral hematoma-induced iron accumulation and neuronal death in piglets. Stroke 40, 2241–2243. Hua, Y., Wu, J., Keep, R.F., Nakamura, T., Hoff, J.T., Xi, G., 2006. Tumor necrosis factor-alpha increases in the brain after intracerebral hemorrhage and thrombin stimulation. Neurosurgery 58, 542–550 (discussion). Ip, J.P., Nocon, A.L., Hofer, M.J., Lim, S.L., Muller, M., Campbell, I.L., 2011. Lipocalin 2 in the central nervous system host response to systemic lipopolysaccharide administration. J. Neuroinflammation 8, 124. Keep, R.F., Hua, Y., Xi, G., 2012. Intracerebral haemorrhage: mechanisms of injury and therapeutic targets. Lancet Neurol. 11, 720–731. Liu, Q., Nilsen-Hamilton, M., 1995. Identification of a new acute phase protein. J. Biol. Chem. 270, 22565–22570. Marques, F., Rodrigues, A.J., Sousa, J.C., Coppola, G., Geschwind, D.H., Sousa, N., Correia-Neves, M., Palha, J.A., 2008. Lipocalin 2 is a choroid plexus acute-phase protein. J. Cereb. Blood Flow Metab. 28, 450–455. Mehdiratta, M., Kumar, S., Hackney, D., Schlaug, G., Selim, M., 2008. Association between serum ferritin level and perihematoma edema volume in patients with spontaneous intracerebral hemorrhage. Stroke 39, 1165–1170. Mucha, M., Skrzypiec, A.E., Schiavon, E., Attwood, B.K., Kucerova, E., Pawlak, R., 2011. Lipocalin-2 controls neuronal excitability and anxiety by regulating dendritic spine formation and maturation. Proc. Nat. Acad. Sci. USA 108, 18436–18441. Naude, P.J., Nyakas, C., Eiden, L.E., Ait-Ali, D., van der Heide, R., Engelborghs, S., Luiten, P.G., De Deyn, P.P., den Boer, J.A., Eisel, U.L., 2012. Lipocalin 2: novel component of proinflammatory signaling in Alzheimer’s disease. FASEB J. 26, 2811–2823.
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Okauchi, M., Hua, Y., Keep, R.F., Morgenstern, L.B., Schallert, T., Xi, G., 2010. Deferoxamine treatment for intracerebral hemorrhage in aged rats: therapeutic time window and optimal duration. Stroke 41, 375–382. Perez de la Ossa, N., Sobrino, T., Silva, Y., Blanco, M., Millan, M., Gomis, M., Agulla, J., Araya, P., Reverte, S., Serena, J., Davalos, A., 2010. Iron-related brain damage in patients with intracerebral hemorrhage. Stroke 41, 810–813. Qureshi, A.I., Mendelow, A.D., Hanley, D.F., 2009. Intracerebral haemorrhage. Lancet 373, 1632–1644. Rathore, K.I., Berard, J.L., Redensek, A., Chierzi, S., Lopez-Vales, R., Santos, M., Akira, S., David, S., 2011. Lipocalin 2 plays an immunomodulatory role and has detrimental effects after spinal cord injury. J. Neurosci. 31, 13412–13419. Rincon, F., Mayer, S.A., 2012. Intracerebral hemorrhage: clinical overview and pathophysiologic concepts. Transl. Stroke Res. 3, S10–S24. Selim, M., Yeatts, S., Goldstein, J.N., Gomes, J., Greenberg, S., Morgenstern, L.B., Schlaug, G., Torbey, M., Waldman, B., Xi, G., Palesch, Y., 2011. Safety and tolerability of deferoxamine
mesylate in patients with acute intracerebral hemorrhage. Stroke 42, 3067–3074. Srinivasan, G., Aitken, J.D., Zhang, B., Carvalho, F.A., Chassaing, B., Shashidharamurthy, R., Borregaard, N., Jones, D.P., Gewirtz, A.T., Vijay-Kumar, M., 2012. Lipocalin 2 deficiency dysregulates iron homeostasis and exacerbates endotoxin-induced sepsis. J. Immunol. 189, 1911–1919. Wu, J., Hua, Y., Keep, R.F., Nakamura, T., Hoff, J.T., Xi, G., 2003. Iron and iron-handling proteins in the brain after intracerebral hemorrhage. Stroke 34, 2964–2969. Xi, G., Keep, R.F., Hua, Y., Xiang, J., Hoff, J.T., 1999. Attenuation of thrombin-induced brain edema by cerebral thrombin preconditioning. Stroke 30, 1247–1255. Xi, G., Keep, R.F., Hoff, J.T., 2006. Mechanisms of brain injury after intracerebral haemorrhage. Lancet Neurol. 5, 53–63. Yang, J., Goetz, D., Li, J.Y., Wang, W., Mori, K., Setlik, D., Du, T., Erdjument-Bromage, H., Tempst, P., Strong, R., Barasch, J., 2002. An iron delivery pathway mediated by a lipocalin. Mol. Cell 10, 1045–1056.
Available online at www.sciencedirect.com
www.elsevier.com/locate/brainres
Research Report
Role of iron in brain lipocalin 2 upregulation after intracerebral hemorrhage in rats Ming Donga,b, Guohua Xia, Richard F. Keepa, Ya Huaa,n a
Deparment of Neurosurgery, University of Michigan, Ann Arbor, MI, USA Department of Neurology, 1st Affiliated Hospital, Jilin University, Changchun, China
b
ar t ic l e in f o
abs tra ct
Article history:
Brain iron overload has a detrimental role in brain injury after intracerebral hemorrhage
Accepted 6 February 2013
(ICH). Lipocalin 2 (LCN2), a siderophore-binding protein, is involved in cellular iron
Available online 15 February 2013
transport. The present study investigated changes in LCN2 expression after ICH and the
Keywords:
role of iron in those changes. Male Sprague-Dawley rats had an intracaudate injection of
Cerebral hemorrhage
autologous blood (ICH) or iron. Control rats received a needle insertion or saline injection.
Lipocalin 2
Some ICH animals were treated with either vehicle or deferoxamine, an iron chelator. Brain
Iron
LCN2 expression was determined by Western blot analysis and immunohistochemistry.
Deferoxamine
Real-time PCR was also used to confirm brain LCN2 mRNA expression. The number of
Immunohistochemistry
LCN2 positive cells was markedly increased in the ipsilateral basal ganglia and cortex after ICH and most LCN2 positive cells were astrocytes. Western blots showed that brain LCN2 levels were higher at days 1, 3 and 7 in the ipsilateral hemisphere after ICH (70 to 80 fold higher than contralateral hemisphere or sham-operated rats at 3 days), and declined to lower levels at day 14. Iron, but not saline injection also caused brain LCN2 upregulation (a more than 100-fold increase). In addition, systemic treatment of deferoxamine reduced ICH-induced LCN2 upregulation (po0.05). These results suggest that iron has a role in brain LCN2 upregulation following ICH. LCN2 upregulation after ICH may be part of the response to clear iron released from the hematoma during clot resolution. & 2013 Elsevier B.V. All rights reserved.
1.
Introduction
Intracerebral hemorrhage (ICH) is a devastating form of stroke with high morbidity and mortality (Qureshi et al., 2009; Rincon and Mayer, 2012). Evidence suggests that ironoverload is involved in ICH-induced brain damage resulting in perihematoma edema, neuronal death and brain atrophy (Keep et al., 2012; Xi et al., 2006). We have demonstrated that
deferoxamine, an iron chelator, reduces ICH-induced brain injury in young and aged rats (Okauchi et al., 2010; Xi et al., 2006), as well as in pigs (Gu et al., 2009). Clinical studies also found that high levels of serum ferritin, an iron storage protein, are independently associated with severe brain edema and poor prognosis in ICH patients (Mehdiratta et al., 2008; Perez de la Ossa et al., 2010), suggesting a role of iron in ICH-induced brain injury (Selim et al., 2011).
Abbreviations: ICH, Intracerebral hemorrhage; LCN2, Lipocalin 2; GFAP, Glial acidic fibrillary protein n Correspondence to: R5018 Biomedical Science Research Building, University of Michigan, 109 Zina Pitcher Place, Ann Arbor, MI 48109-2200, USA. Fax: þ1 734 763 7322. E-mail address: [email protected] (Y. Hua). 0006-8993/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.brainres.2013.02.008
87
brain research 1505 (2013) 86–92
Lipocalin 2 (LCN2), also known as neutrophil gelatinase associated lipocalin (NGAL) or 24p3, is a siderophore-binding protein. LCN2 mediates iron uptake in cells expressing the LCN2 cell surface receptor, LCN2R (24p3R) (Devireddy et al., 2005). Recently, the function of LCN2 within the CNS has started to be elucidated. In vivo studies have demonstrated LCN2 expression in the choroid plexus in response to peripheral lipopolysaccharide administration (Marques et al., 2008) and excitotoxic brain injury (Chia et al., 2011). Recent studies showed that LCN2 plays a detrimental role in the CNS after spinal cord injury (Rathore et al., 2011) but it appears to play a protective role in experimental autoimmune encephalomyelitis (Berard et al., 2012). In the present study, we examined the time course of brain LCN2 expression in a rat model of ICH. The role of iron in ICH-induced LCN2 expression was also examined.
2.
Results
ICH-induced changes in LCN2 were measured by immunohistochemistry and Western blotting. LCN2 positive cells were found in the ipsilateral basal ganglia but not the contralateral basal ganglia at days 1, 3, 7 and 14 after ICH (Fig. 1A). The LCN2 protein levels determined by Western blotting in the ipsilateral basal ganglia were significantly increased at day 1, remained at high levels at days 3 and 7, and the decreased markedly at day 14 after ICH (Fig. 1B). Similar results were found in the ipsilateral cortex following ICH (Fig. 1C). LCN2 immunoreactivity was very weak in sham-operated brain but increased significantly after blood injection. LCN2 positive cells were astrocyte-like (Fig. 2A). Western blots, with
day 1
LCN2 normalized to b-actin, indicated that LCN2 protein levels in the ipsilateral basal ganglia were 71-fold higher than those in the contralateral basal ganglia (LCN2/b-actin ratio: 2.7070.46 vs. 0.0470.01, po0.001) and 84-fold higher than in sham-operated animals (0.9270.14 vs. 0.0170.01, po0.001) at 3 days after ICH (Fig. 2B). Similar results were also found in the cortex (Fig. 2B). Double-labeling was used to determine which types of cells express LCN2 after ICH. We found that LCN2 positive cells predominantly colocalized with glial fibrillary acid protein (GFAP) positive cells after ICH. There were only a few LCN2 positive cells that colocalized with neuronal nuclear antigen (NeuN) in cortex (Fig. 3). These results suggest that LCN2 is mainly expressed in astrocytes following ICH. To test whether iron can induce LCN2 expression, 30 mL of ferrous chloride (1 mmol/L) or saline was injected into the rats’ right basal ganglia. There were many more LCN2 positive cells in the ipsilateral basal ganglia after iron injection compared to saline injection (Fig. 4A). Iron injection caused a 136-fold increase of LCN2 protein levels at day 3 (ratio of LCN2/b-actin: 1.7770.43 vs. 0.01370.004 in saline injection, po0.01, Fig. 4B). To examine whether ICH-induced LCN2 expression can be attenuated by iron chelation, rats were treated with deferoxamine or vehicle (2 h and then every 12 h for 7 days) after ICH. Deferoxamine reduced ICH-induced LCN2 upregulation at day 7 (ratio of LCN2/b-actin: 0.4470.23 vs. 1.7370.5 in vehicle, po0.05, Fig. 5). To further elucidate whether the brain produces LCN2 after ICH, quantitative real-time PCR of LCN2 gene expression was examined in triplicate using Eppendorf Mastercycler Realplex Detection System. Real-time PCR showed that LCN2 mRNA expression in the ipsilateral basal ganglia was increased 6.5-fold compared to the contralateral basal ganglia at 24 h after ICH (po0.05).
day 3
day 7
day 14
Ipsilateral basal ganglia * Contralateral basal ganglia
Ipsilateral basal ganglia day1
day3
day7
Ipsilateral cortex day1
day14
LCN2
LCN2
β−actin
β−actin
day3
day7
day14
Fig. 1 – Time course showing LCN2 immunoreactivity in the ipsi- and contra-lateral basal ganglia (A), and the protein levels of LCN2 and b-actin in the ipsilateral basal ganglia (B) and cortex (C) after an injection of 100 ll autoglous blood into the right caudate. The asterisk indicates the hematoma, the boxes indicate the locations where higher powered immunohistochemistry images were taken. Scale bar¼ 100 lm.
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ICH Sham
Contraleteral
Ipsilateral
Basal ganglia
Cortex
Bar =100μm
ICH Basal ganglia
Ipsilateral
ICH
Sham
ICH
Sham
LCN2
LCN2
β−actin
β−actin ICH Ipsilateral
Cortex
Contralateral
Contralateral
LCN2
LCN2
β−actin
β−actin
Fig. 2 – LCN2 immunoreactivity in the ipsilateral basal ganglia and cortex after a sham operation, and in the contra- and ipsilateral basal ganglia and cortex after ICH at day 3(A), scale bar¼ 100 lm. The protein levels of LCN2 and b-actin determined by Western blotting in the basal ganglia and cortex (B) 3 days after sham operation or blood injection.
LCN2
GFAP
Merge
LCN2
NeuN
Merge
Fig. 3 – LCN2, glial fibrillary acidic protein (GFAP) and NeuN immunoreactivity and the colocalization of LCN2 with GFAP or NeuN by double labeling in the ipsilateral basal ganglia at day 3 after 100 ll blood infused into the right basal ganglia. Scale bar¼ 50 lm.
3.
Discussion
The major findings of the present study are: (1) brain LCN2 levels are markedly increased after ICH; (2) most LCN2 positive cells after ICH are astrocytes; (3) iron can upregulate brain LCN2; and (4) deferoxamine reduces ICH-induced LCN2
upregulation. These findings suggest an important role of iron in brain LCN2 upregulation after ICH and potentially a role of LCN2 in handling iron that is released from the hematoma after ICH. LCN2 expression in the brain is prominently increased at an early stage of ICH, which may be associated with acute
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Iron injection
Saline injection
Basal ganglia
Cortex
Saline injection
Iron injection
Basal ganglia
β−actin
Fig. 4 – LCN2 immunoreactivity in the ipsilateral basal ganglia and cortex, and the protein levels of LCN2 and b-actin in the ipsilateral basal ganglia at day 3 after 30 ll of ferrous iron (1.0 mmol/L) or saline injection into the rat basal ganglia. Scale bar¼ 100 lm.
ICH+Vehicle
ICH+DFX
LCN2
ICH+Vehicle
ICH+DFX
LCN2
β-actin
Fig. 5 – (A) LCN2 immunohistochemistry of the ipsilateral basal ganglia, and (B) Western blot of that tissue from rats treated with deferoxamine (DFX) or vehicle for 7 days after 100 ll blood was injected into the right caudate. Scale bar¼100 lm.
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brain injury. There was a very marked increase in LCN2 in the perihematomal zone of basal ganglion and cortex occurs 1 day after ICH. Brain LCN2 levels were high for 7 days and decreased around 14 days after ICH. Although levels of LCN2 mRNA and protein expression are low in most regions of the normal brain (Chia et al., 2011), LCN2 is rapidly inducible (Liu and Nilsen-Hamilton, 1995). Recent work showed that LCN2 expression can be induced in the central nervous system following injury and neuroinflammation (Berard et al., 2012; Chia et al., 2011; Ip et al., 2011). Our study also confirmed that the brain can produce LCN2. Many factors may cause brain LCN2 upregulation after ICH. Our results showed that intracerebral injection of iron increases brain LCN2 levels and deferoxamine, an iron chelator, reduces ICH-induced upregulation of brain LCN2. These results indicate a role of iron in regulating LCN2 expression following ICH. However, LCN2 is an acute phase protein and other factors, including tumor necrosis factoralpha (TNF-a) and thrombin, two major factors which cause brain injury after ICH (Bodmer et al., 2012), may also upregulate brain LCN2 in the acute phase. For example, TNF-a is increased in the brain two hours after ICH (Hua et al., 2006) and TNF-a can upregulate LCN2 (Naude et al., 2012). Future studies should determine whether thrombin has a role in ICH-induced upregulation of brain LCN2. The role of LCN2 in ICH is unclear, but LCN2 upregulation in the brain after ICH may affect iron homeostasis. It is well known that iron overload following ICH causes brain damage. LCN2 transfers iron from intracellular to the extracellular medium via binding with intracellular siderophore, thereby reducing intracellular iron concentration. A report has suggested that LCN2 could be a mediator of an alternative, transferrin-independent pathway for cellular iron delivery (Yang et al., 2002). The iron chelator, deferoxamine, protects endotoxin-induced toxicity and mortality in LCN2 deficient mice (Srinivasan et al., 2012). Future studies should determine whether LCN2 can enhance brain iron clearance following ICH. We observed that LCN2 was mainly expressed in astrocytes after ICH. Our results are supported by other studies. In the case of kainite-induced brain injury, LCN2 is highly expressed in the reactive astrocytes in the injured hippocampus (Chia et al., 2011). Similarly, after spinal cord contusion injury, Rathore et al. (2011) found a strong induction of LCN2 in astrocytes but not microglia. After ICH, the breakdown of hemoglobin in the hematoma results in the release of iron. Thus, there is an increase in perihematomal iron, that can be detected by Perls’ staining, which persists for weeks and is mostly present in glial cells (Wu et al., 2003). LCN2 upregulation in perihematomal astrocytes suggests that it may be involved in, or a response to, iron accumulation. We also found some expression of LCN2 in neurons after ICH. LCN2 is also found to be expressed in neurons and upregulated in the hippocampus by stress (Mucha et al., 2011). Neuronal expression of LCN2 has been observed following exposure to trauma (Rathore et al., 2011) or neuroinflammation (Berard et al., 2012; Chia et al., 2011; Ip et al., 2011). After ICH, Perls’ staining indicates that there is a build-up of iron in some neurons (Wu et al., 2003). Whether LCN2 participates in that uptake and in the neuronal injury that follows ICH deserves further investigation.
In conclusion, LCN2 is markedly upregulated in the brain after ICH. As an intracerebral iron injection induces brain LCN2 and iron chelation reduces ICH-induced LCN2 upregulation, iron appears to play a central role in the LCN2 response to hemorrhage. LCN2 may be involved in iron homeostasis after cerebral hemorrhage and modifying brain LCN2 levels may affect ICH-induced brain iron overload and brain injury.
4.
Experimental procedures
4.1.
Animal preparation and intracerebral injection
Animal use protocols were approved by the University of Michigan Committee on the Use and Care of Animals. A total of 96 male Sprague-Dawley rats (weighed 275–350 g, Charles River Laboratories, Portage, MI, USA) were used in these studies. Animals were housed under standard 12:12 h light–dark conditions and allowed free access to food and water. Animals were anesthetized with pentobarbital (45 mg/kg, i.p.). Septic precautions were used in all surgical procedures and rectal temperature was maintained at 37.5 1C using a feedback-controlled heating pad. A polyethylene catheter (PE-50) was inserted into the right femoral artery to monitor arterial blood pressure and blood gases and to obtain blood for intracerebral infusion. The animals were positioned in a stereotactic frame (Kopf Instruments) and a cranial burr hole (1 mm) was drilled. Rats received an autologous blood injection into the right basal ganglia (coordinates: 0.2 mm anterior, 3.5 mm lateral, and 5.5 mm ventral to the bregma) through a 26 g needle at a rate of 10 mL/min using a microinfusion pump.
4.2.
Experimental groups
This study was divided into four parts. In the first part, rats received either an intracerebral injection of 100 mL autologous whole blood or a needle insertion (sham). Rats were euthanized 1, 3, 7, or 14 days later for Western blot analysis (n¼ 4 for each time point) and immunohistochemistry (n¼ 4 for each time point). In the second part, rats had an infusion of 30 mL ferrous chloride (1 mmol/L) or saline. Rat brains were sampled at 3 days after intracerebral injection and used for Western blot analysis (n ¼4) and immunohistochemistry (n ¼4). In the third part, rats had an intracerebral infusion of 100 mL autologous whole blood and were treated with either deferoxamine (100 mg/kg, i.p. at 2 h after ICH and then every 12 h for 7 days) or vehicle. Rats were euthanized at day 7 for Western blot analysis (n¼ 4) and immunohistochemistry (n ¼4). In the fourth part, rats had a 100 ml blood injection in the right basal ganglia and were killed at 24 h for RNA isolation and real-time PCR analysis.
4.3.
Western blot analysis
Western blot analysis was performed as previously described (Xi et al., 1999). Briefly, brain tissue was immersed in Western sample buffer and sonicated. Protein concentration was determined by Bio-Rad protein assay kit and 50 mg protein samples were separated by sodium dodecyl sulfate–polyacrylamide gel
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electrophoresis and transferred to a Hybond-C pure nitrocellulose membrane (Amersham). Membranes were probed with primary antibody: goat anti-lipocalin 2 antibody (1:200 dilution, R&D Systems). The secondary antibody was rabbit anti-goat IgG (Bio-Rad; 1:2500 dilution). The antigen–antibody complexes were visualized with the ECL chemiluminescence system (Amersham) and exposed to Kodak X-OMAT film. The relative densities of bands were analyzed with NIH ImageJ.
4.7.
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Statistical analysis
All data in this study are presented as mean7SD. Data were analyzed with Student’s t-test and analysis of variance. The P value of less than 0.05 was considered as significant level.
Acknowledgment 4.4.
Immunohistochemistry
Immunohistochemistry was performed as described previously (Xi et al., 1999). Briefly, rats were anesthetized and subjected to intracardiac perfusion with 4% paraformaldehyde in 0.1 mol/L phosphate-buffered saline (pH 7.4). Brains were removed and kept in 4% paraformaldehyde for 6 h, then immersed in 30% sucrose for 3–4 days at 4 1C. After embedding in a mixture of 30% sucrose and OCT (SAKURA Finetek, USA), 18 mm sections were taken on a cryostat. Immunohistochemistry staining was then performed using the avidin–biotin complex technique. The primary antibody was goat anti-lipocalin 2 antibody (1:200 dilution, B&D Systems). The secondary antibody was rabbit anti-goat IgG (Bio-Rad; 1:2500 dilution).
4.5.
Immunofluorescent double-labeling
For immunofluorescent double-labeling, the primary antibodies were goat anti-lipocalin 2 (1:200 dilution, R&D Systems), mouse anti-NeuN (1:200 dilution, Millipore), and mouse antiGFAP (1:200 dilution, Millipore). Rhodamine-conjugated donkey anti-goat (Invitrogen; 1:500 dilution), fluorescein isothiocyanate (FITC)-labeled horse anti-mouse (Vector; 1:500 dilution) and donkey anti-rabbit antibodies were used as secondary antibodies. The double-labeling was analyzed using a fluorescence microscope (Xi et al., 1999).
4.6. Quantitative real-time polymerase chain reaction (qPCR) Total RNA was prepared from 20–30 mg the ipsilateral basal ganglia tissue using an RNeasy Mini kit (Qiagen), and stored at 80 1C. 2 mg mRNA was used for cDNA synthesis using High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA). Quantitative real-time PCR of gene expression were run in triplicate using TaqMan PCR Master Mix (Roche). No template controls were prepared by omitting to add cDNA template to the reaction. Real-time quantitative primers were designed and purchased from Applied Biosystems (Life Technologies Corporation). The rat LCN2 primers were 50 -GAT TCG TCA GCT TTG CCA AGT-30 (forward primer) and 50 -CAT TGG TCG GTG GGA ACA G-30 (reverse primer). A positive standard curve for each primer was obtained using serially-diluted cDNA sample mixture. The quantity of gene expression was calculated using standard samples and normalized with GAPDH.
This study was supported by Grants NS-039866, NS-057539, NS-073595 and NS-079157 from the National Institutes of Health (NIH). The content is solely the responsibility of the authors and does not necessarily represent official views of the NIH.
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